Nowadays, the sequences of the basic building blocks of biopolymers and their post-translational modifications (PTM) are identified predominantly with the aid of tandem mass spectrometers. In the following, peptides and proteins as biopolymers are dealt with in particular, although the invention should not be limited to these. For example, oligosaccharides are a further group of biopolymers whose monomeric basic units, the sugar molecules, can be linked with each other in linear or in branched form. A key technology for these investigations is the fragmentation of the biopolymer ions in the mass spectrometer. There are two fundamentally different types of fragmentation: ergodic fragmentation and non-ergodic, electron-induced fragmentation, for each of which several methods are known. The electron-induced fragmentation of the peptide or protein ions is complementary to the ergodic fragmentation, firstly because it cleaves at different points of the amino acids within the chain of amino acids, and secondly because it does not remove the side chains of the post-translational modifications during fragmentation, as is the case with ergodic fragmentation. By comparing the fragment ion spectra obtained ergodically with those obtained non-ergodically, the sequences as well as the mass and position of the modifications can be read.
A relatively simple electron-induced fragmentation method is electron transfer dissociation. It is possible to use specific types of negative reactant ions to cleave multiply positively charged biopolymer ions, particularly peptide or protein ions, by the transfer of an electron (“ETD”=electron transfer dissociation). The reactant ions are usually radical anions of the form M•− of a molecule M; these radical anions easily give up electrons. See U.S. Published Patent Application 2005/0199804 A1 (D. F. Hunt et al.) and German Patent DE 10 2005 004 324 B4 (R. Hartmer and A. Brekenfeld). Both documents describe how multiply charged positive peptide or protein ions are fragmented by reactant ions with this method. Electron transfer dissociation is a special form of the general group of charge transfer reactions.
A second form of charge transfer reaction which is often used is the reaction between multiply positively charged analyte ions and non-radical negatively charged ions of the form (M−H)− or (M+H)−, which can be used to reduce the number of respective charges on the positive analyte ions (“PTR”=proton transfer reactions, also called “charge stripping”). In favorable cases, the non-radical anions required for this can be obtained in electron attachment ion sources from the same substances that are used for the production of radical anions for ETD (see U.S. Pat. No. 7,582,862 B2) by changing the operating conditions. The charge reduction enables highly charged analyte ions to be converted into less highly charged ions in order to reduce the complexity of the mass spectra from mixtures of many different highly charged analyte ions and to produce favorable biopolymer ions for electron transfer dissociation.
The reactions for electron transfer dissociation (ETD) and also for charge reduction by proton transfer (PTR) take place in reaction cells, in which both positive and negative ions can be stored. These can be two-dimensional RF ion traps with pseudopotential barriers at the ends, for example, but also three-dimensional RF ion traps. These reaction cells are usually filled with a damping gas, in which the ion motions are thermalized. Mass spectrometers with both types of reaction cells are commercially available and are known to those skilled in the art. The positive analyte ions and the negative reactant ions are generally introduced sequentially into the ion traps and mixed there. The reactions can then occur without any further assistance.
U.S. Published Patent Application 2005/0199804 (“'804 Application”) explains that substances for the formation of ETD reactant ions can be found in the group of polycyclic aromatic hydrocarbons (polyaromatic hydrocarbons). Specifically, the substances anthracene, naphthalene, fluorene, phenanthrene, pyrene, fluoranthene, chrysene, triphenylene, perylene, acridine and others are named. With some of these substances, however, the electron attachment ion source always supplies non-radical anions of the form (M−H)− also, as can be seen in Table 1 of the '804 Application, and these produce undesirable proton transfer reactions here. Thus not all of these polycyclic aromatic compounds are equally advantageous for ETD. Disadvantageous, on the whole, for all polycyclic aromatic compounds is the fact that they have very low vapor pressures. To avoid condensation in relatively cool supply lines, it is therefore necessary to keep these substances in a heated vessel close to the electron attachment ion source, which must also be heated, in the interior of the mass spectrometer. This makes refilling complicated, and may even have to be done by the manufacturer's service staff. Although it would be possible to heat all the supply lines between a heated vessel external to the vacuum system and the electron attachment ion source, it is extremely difficult to uniformly heat supply lines that pass through the wall of the vacuum system.
High ETD effectiveness of the anions of a substance means here that, on the one hand, a high yield of fragment ions is achieved and, on the other hand, no significant proportion of proton-transfer reactions occurs. According to the '804 Application, fluoranthene is particularly ETD effective in this sense.
Published U.S. Patent Application U.S. 2010/0140466 A1, incorporated herein by reference, proposes aliphatic substances with electron affinities between 0.3 and 0.8 electron volts, particularly 1,3,5,7-cyclooctatetraene (m=104.15 Da, EA=0.550 eV); trichloroethene, (m=131.39 Da; EA=0.400 eV), tetrachloroethene (m=165.83 Da; EA=0.64 eV) and 2,3-butanedione (m=86.09 Da; EA=0.69 eV). The advantage of these substances is that their higher vapor pressures mean that they can be kept in an unheated vessel external to the mass spectrometer, which facilitates the refilling. 1,3,5,7-cyclooctatetraene, which was preferred initially, has turned out to be not particularly suitable for some types of reaction cells because in these cells it very quickly loses the electrons, which are only weakly bound. As the best substance in this group, 2,3-butanedione provides good yields of fragment ions of the multiply positively charged analyte ions. The ETD effectiveness approaches that of fluoranthene.
2,3-butanedione also has disadvantages, however. Firstly, it is a respiratory and eye irritant with hotly debated toxicity (approved as butter flavor in Europe, although it is known to cause pulmonary diseases), and therefore its use is neither simple nor without risk, precisely because of its otherwise favorable vapor pressure. Secondly, it has a very low molecular mass of only 86 atomic mass units. If it is to be stored in an RF storage cell, the RF voltage must be lowered to such an extent that heavy positive ions are lost because of the limited mass range of all such storage cells. And thirdly, the electron attachment ion source which is used to produce the anions from butanedione cannot be switched to also produce the non-radical anions which can be used for proton-transfer reactions (PTR) for the charge reduction. The search for further, favorable substances for the ETD is thus not concluded.
PCT Application WO 2011/092515 discloses substances with Franck-Condon factors between 0.1 and 1.0, and electron affinities between 0.1 and 150 kJ/mol (1.55 eV). A list of more than 90 substances is provided, starting with 1,3- and 1,4-dicyanobenzol. The list contains both aromatic and aliphatic compounds from very different substance classes.
The electron affinity (EA) is the energy which must be used to remove the electron from the radical anion again, i.e., the binding energy of the added electron. This binding energy must not be too low because, otherwise, the substance hardly accepts any electrons, on the one hand, and, having accepted an electron, the anions easily lose it again, on the other hand. The binding energy must not be too high either, however, because otherwise the positive biopolymer ions cannot detach and attract the electron, i.e., cannot affect the electron transfer.
The aliphatic ETD substances listed above were taken from the extensive NIST database for organic substances (webbook.nist.gov), which represents one of the most complete databases for physical chemistry data of organic chemical substances. In its table of organic substances with known electron affinity, only around 200 substances in total are listed in the range from EA=0.3 to 0.8 eV, and few of them are suitable aliphatic compounds, whereas nearly all the polycyclic aromatic compounds from the '804 Application can be found there. It must be assumed that for a large majority of organic substances, especially aliphatic substances, the electron affinities are not known, so a search in the specialist literature promises little success.
There is a need of providing starting substances for the production of anions for charge transfer reactions, particularly substances for the production of radical anions for electron transfer dissociation with high ETD efficiency which do not have the disadvantages observed with the ETD reagents known up to now.